ALBERT

Description: Thrombopoietin (TPO) is a 332 amino acid cytokine that plays a key role in the regulation of megakaryopoiesis and platelet production. Over the past 10 years, recombinant forms of TPO have shown various degrees of effectiveness in the treatment of thrombocytopenias associated with chemotherapy or various disease states, although therapy with these agents may be associated with a risk of producing neutralizing antibodies. A small-molecule nonpeptidyl TPO receptor agonist is expected to offer a safer alternative to these protein agents and also offer advantages in terms of lower cost of production and an easier oral route of administration. The mode of action of TPO involves oligomerization of its specific cell-surface receptor followed by activation of multiple signaling proteins such as the JAK1/JAK2 kinases and the STAT transcription factors. Using a high-throughput reporter-gene assay based on activation of STATs in BAF-3/TPO-R cells, screening of a library of 260,000 small-molecule compounds resulted in the identification of diazo naphthalenesulfonic acids as a novel series of TPO-R agonists. Modification of the core structure and adjustment of unwanted functionality resulted in the development of hydrazino naphthalenesulfonates which displayed efficacies equivalent to those of TPO in several cell-based assays, such as luciferase reporter gene and proliferation in a TPO receptor-dependent way (i.e., no effect on TPO-insensitive cell lines). Furthermore, these compounds elicited signal-transduction responses, such as JAK2 and STAT-5 activation, in TPO-receptor-expressing cells similar to those in TPO itself, and promoted megakaryocyte differentiation in cultures of human bone marrow cells. Potencies for the best compounds in the series were quite high for such small molecules, with EC50 values in the 10–100 nM range. However, the compounds were devoid of oral bioavailability, presumably due to the highly polar sulfonic acid functionality. Molecular modeling studies of three different series of agonists suggested a number of potential replacements of the sulfonate group, which resulted in the development of the biphenyl carboxylates, a series of compounds that showed excellent pharmacokinetic parameters, including oral bioavailability. Out of a total of over two hundred analogs, SB-497115 was selected as the candidate for clinical studies due to its optimum biological and PK properties: the compound showed full maximal efficacy of TPO both in the proliferation of BAF-3/TPO-R cells (EC50 = 30 nM) as well as in the increase of the number of CD41+ cells, a marker of megakaryocyte differentiation, in cultures of human bone marrow cells (EC50 = 100 nM). Oral bioavailabity of SB-497115 was determined to be 26%, 83% and 89%, for rat, dog and monkey, respectively. The identification of SB-497115 (MW of 442) as a TPO mimetic provides proof of principle for drug discovery using JAK/STAT based assays, and shows for the first time that a small non-peptidyl molecule can trigger the selective activation of a cytokine receptor which is confirmed by the elevation in platelet numbers in human volunteers, as demonstrated in Phase I clinical studies. Evaluation of the potential of SB-497115 as an agent for the treatment of thrombocytopenia in humans is ongoing.

Description: SB-497115 is a selective, low molecular weight, non-peptidyl thrombopoietin receptor (TpoR) agonist. identified by its ability to activate the JAK/STAT signalling pathway. SB-497115 is being developed for the treatment of thrombocytopenias, such ass, immune thrombocytopenic purpura and chemotherapy-induced thrombocytopenia. SB-497115 requires TpoR to activate the JAK/STAT signalling pathway and stimulates transcription through the STAT based (IRF-1) and megakaryocyte specific (gpIIb) promoters. An analysis of the receptor selectivity of SB-497115 was undertaken utilizing a panel of various transfected and non-transfected cell lines in which other cytokines, including G-CSF, Epo, IL-3, Interferon -alpha or Interferon-gamma, were active. SB-497115 was inactive over a three-fold concentration range in proliferation, reporter gene, or STAT activation assays performed on cell lines that did not express TpoR. To characterize the kinetics and specificity of SB-497115 in cells, multiple molecular markers for Tpo activity were measured. Western blot analysis for activation of the STAT and MAPK pathways was performed using phospho-specific antibodies on lysates of UT7-Tpo cells treated with SB-497115. In addition, mRNA expression of several early response genes associated with proliferation and Tpo activation (i.e., Fos, EGR-1 and thyroid-like receptor 3 ), was measured in response to SB-497115 treatment. The kinetics and level of induction for pathway phosphorylation events and gene expression were similar to that seen with Tpo. A proliferative response in the human Tpo-dependent cell line, UT7-Tpo, by SB-497115 was assayed by thymidine incorporation and an EC50 of 30 nM was demonstrated. An analysis of the receptor selectivity of SB 497115 was undertaken utilizing a panel of various transfected and non-transfected cell lines in which other cytokines, including G-CSF, Epo, IL-3, Interferon-alpha or Interferon-gamma, were active. SB 497115 demonstrated a complete lack of activity over a three-fold concentration range in proliferation, reporter gene or STAT activation assays performed on cell lines that did not express TpoR. SB-497115 was shown to be equal to or better than rhTpo in the ability to induce differentiation of normal human bone marrow progenitors (CD34) into CD41+ cells of the megakaryocyte lineage, with an EC50 of 100 nM. These latter activities of SB-497115 were similar to or greater than the level of maximal activity seen with Tpo in these assays. SB-497115 demonstrates specificity for human and chimpanzee TpoR and increases platelet counts in both chimpanzees and normal human volunteers when dosed orally. In conclusion, SB-497115 is the first non-peptide small molecule TpoR agonist to demonstrate activity in human in vitro bone marrow assays and demonstrate pharmacological activity in humans.